Rubella virus is the only member of the Rubivirus genus within the Togaviridae family. The small enveloped (+) RNA virus is a human pathogen and causes a mild, self-limiting childhood disease (German measles or rubella) characterized by rash, lymphadenopathy and low-grade fever. When acquired in the first trimester of pregnancy, however, it may cause stillbirth, spontaneous abortion or several anomalies associated with the congenital rubella syndrome. The characteristic triad of congenital rubella syndrome includes cataracts, heart defects and deafness of the fetus. It necessitates rubella vaccination programs and surveillance of the immune status of women in child-bearing age.
The structural proteins of the rubella virus originate from a single 110 kDa polypeptide precursor, which is proteolytically cleaved to yield the capsid protein C and the envelope proteins E2 and E1. E2 and E1 are glycosylated, they form non-covalent heterodimers at the surface of the virion and are the preferred targets of the humoral immune response. The membrane-anchored ectodomain of the E1 protein, in particular, is immunodominant, and antibodies against E1 are abundant in sera from rubella-infected individuals.
The rubella E1 protein, also termed rubella hemagglutinin (see FIG. 1), presumably consists of a large ectodomain (residues 1-452), followed by a single transmembrane helix (residues 453-468) and a short cytoplasmic tail (residues 469-481). The residues 438-452, which immediately precede the transmembrane region, probably form also a helix. The ectodomain of E1 bears 20 cysteine residues, which are engaged in ten disulfide bonds. The cysteine pairs C(1)-C(2), C(3)-C(15), C(6)-C(7), C(9)C(10), C(11)-C(12), C(13)-C(14), C(17)-C(18) and C(19)-C(20) could be confirmed with certainty, whereas the pairing of the cysteine residues C(4), C(5), C(8) and C(16) remains ambiguous (Gros et al. 1997, Virology 230, 179-186). The ectodomain is glycosylated at the three asparagines 76, 177 and 209.
There have been several attempts in prior art to produce the rubella E1 protein for diagnostic purposes. Initially, soluble fragments of E1 to be used as antigens for immunoassays were isolated from the supernatant of infected Baby hamster kidney (BHK-21) or Vero cells. Later, various expression and secretion systems were developed with the aim of producing soluble and immunoreactive versions of E1 in eukaryotic hosts (Hobman et al. 1994, Virus Res. 31, 277-289 and Seto et al. 1994, J. Med. Virol. 44, 192-199). A glycosylated and soluble form of full-length E1 could be produced in baculovirus-infected Spodoptera frugiperda (Seppänen et al. 1991, J. Clin. Microbiol. 29, 1877-1882 and Oker-Blom 1989, Virology 172, 82-91) and CHO cells (Perrenoud et al. 2004, Vaccine 23, 480-488) and, most recently, in Pichia pastoris (Wen and Wang 2005, Intervirology 48, 321-328). The expression of rubella-like particles in BHK cells (Grangeot-Keros et al. J. lin. Microbiol. 33, 2392-2394) and in a stably transfected CHO cell line (Giessauf et al. 2005, Arch. Virol. 150, 2077-2090) yielded rubella antigens suitable for diagnostic purposes. These rubella-like particles are non-infectious, ill-defined agglomerates of the covalently linked rubella proteins C, E2 and E1, and are useful for detecting immunoglobulins of the M and G type.
Non-glycosylated forms of E1 could, in principle, be produced much more efficiently in a prokaryotic host. In an early attempt, a full-length and a truncated version (207-353) of rubella E1 were fused to protein A from Staphylococcus aureus and produced in E. coli (Terry et al. 1989, Arch. Virol. 104, 63-75). These fusion proteins were active as antigens, but not well soluble and therefore only of limited value for the specific detection of anti-E1 antibodies. In general, variants of E1 from prokaryotic hosts showed a strong tendency to aggregate, possibly because they are unglycosylated, or because they are incorrectly disulfide-bonded. In fusion with glutathione-S-transferase, only small fragments of E1 comprising as little as 75 or 44 amino acid residues could be expressed in a soluble and functional form (Newcombe et al. 1994, Clinical and Diagnostic Virology 2, 149-163 and Starkey et al. 1995, J. Clin. Microbiol. 33, 270-274). Larger E1 fragments encompassing 82 or 171 amino acid residues could be obtained when fused to both RecA and β-galactosidase (Wolinsky et al. 1991, J. Virol. 65, 3986-3994).
The oxidative refolding of large cysteine-rich proteins such as E1 is very difficult, because misfolded intermediates with wrong disulfides, which are trapped during refolding, have a very high tendency to aggregate. Therefore, many efforts concentrated on finding contiguous B-cell epitopes along the E1 polypeptide chain and to use corresponding short soluble peptides as antigens in immunoassays. Antibodies generally show modest affinities towards small peptide antigens, and therefore it remains a major aim to produce stable and soluble fragments of E1 with a high antigenicity and in high amounts, ideally by the massive production as inclusion bodies in a prokaryotic host, followed by a robust renaturation procedure.
In Newcombe et al., (supra) glutathione-S-transferase (GST) E1 fusion proteins were used to produce rubella E1 antigen fragments in E. coli in a soluble form. However, only after a substantial truncation of the E1 sequence a soluble expression was feasible for the cysteine-free region 243-286 (44 amino acid residues). European Patent Application EP-A-0299673 discloses a peptide from amino acid residues 207-353 which retains rubella Ig specific binding characteristics.
Furthermore, Starkey et al., (supra) disclose that a very short segment of 44 to 75 amino acid residues of rubella E1 was soluble when fused to GST. GST fusion proteins containing the entire E1 sequence as well as large E1 subfragments were expressed as insoluble inclusion bodies which could neither be purified nor renatured and were therefore discarded.
European patent application No. EP-A-1780282 discloses the recombinant expression and production of soluble rubella E1 envelope antigens that are characterized by lacking at least the C-terminal transmembrane region and the anchor segment as well as at least the segment from amino acids 143 to 164 in the middle part of the molecule. These rubella E1 antigens contain at least the region spanning the disulfide bridges Cys 349-Cys 352 and Cys 368-Cys 401 and optionally Cys 225-Cys 235. According to the teaching of EP-A-1782082 it is essential to have both disulfide bridges in the C-terminal part of the antigen intact, i.e., closed, to obtain a rubella E1 variant that is sufficiently antigenic and suitable for the detection of antibodies against rubella virus in a sample.
The problem to be solved was therefore to generate soluble rubella E1 variants which harbor further combinations of disulfide-stabilized epitopes and which are highly soluble and highly reactive in terms of immunology (i.e. highly antigenic), and therefore well-suited as antigens for diagnostic applications.